Project Summary Hirsch sprung disease (HSCR) is a congenital absence of neurons in a portion of the intestinal tract, due to a failure of neural cres cell (NCC) colonization or development. This anomaly occurs in 1/5000 live births, and typically requires surgical resection of the a ganglionic bowel. Only half of the HSCR cases are attributable to known genetic defects, and it is therefore necessary to explore other pathways and potential modifiers of this disease. The proposed studies investigate the role of vitamin A metabolism in NCC and gastrointestinal tract development using new animal models of retinoid deficiency. I will test the hypothesis that RDH10 has a significant impact on the RA synthesis necessary for organogenesis, specifically on NCC proliferation, differentiation or migration during formation of the enteric nervous system (ENS). Specifically, this project addresses the effects of retinoid deficiency in an allelic series of mice as well as investigates the cause and potential rescue of HSCR. Aim 1 will define the temporal and spatial requirements for retinoid signaling during gut development, testing our hypothesis that RA signaling is spatiotemporally required for proper ENS formation. This is relevant, as Vitamin A deficiency can occur prior to and during pregnancy and can affect NCC development, resulting in congenital anomalies that may include HSCR. Hence it is critical to understand which stage of NCC development requires retinoid signaling during ENS formation and gut development. RDH10 is a retinol dehydrogenase enzyme required for the first oxidation step of vitamin A to retinoic acid (RA). We hypothesize in Aim 2 that RDH10 is required for the initial migration of neural crest cells, and without proper RA signaling, NCCs do not receive the appropriate guidance signals required to colonize the gut and form a normal ENS. We utilize retinoid supplementation both in culture and via in utero gavage supplementation, along with conditional knockout mice to define the temporal requirement for RA signaling. Our results will act as a springboard for understanding the cellular and molecular mechanisms underlying defective ENS development in RDH10-deficient mice, as well as determine the interactions of RA on known enteric NCC regulatory pathways and gut microenvironment. We will use immunohistochemistry and in situ hybridization on whole embryos, guts, cultured guts, and sections to assay for enteric NCC proliferation, apoptosis, migration, and differentiation, as well as for genes known to contribute to these developmental processes, alongside a combination of candidate gene and RNA sequencing approaches. Results from these studies will establish a new model for studying HSCR, providing information regarding the spatiotemporal requirements for RA during embryonic intestinal development, the pathogenic cellular mechanisms of RDH10 deficient animals, as well as the interactions of RA with pathways that govern normal ENS NCC colonization. This knowledge may lead to innovative non-surgical treatments to reduce the morbidity and mortality of this common congenital disease.